Wound contraction inhibitor

ABSTRACT

The pharmaceutical formulation for inhibiting wound contraction of the present invention includes secretory leukocyte protease inhibitor as an active ingredient. The SLPI concentration in the pharmaceutical formulation is preferably 1–5000 ng/ml, and more preferably 1–100 ng/ml. The pharmaceutical formulation for inhibiting wound contraction of the present invention may be prepared as an external preparation including a base and the SLPI as an active ingredient. The pharmaceutical formulation for inhibiting wound contraction of the present invention may be prepared as an injection including the SLPI as an active ingredient. The external preparation may preferably include a preservative. The injection may preferably include a stabilizer (an antioxidant), a preservative and an analgesic agent.

BACKGROUND OF THE INVENTION

The present invention relates to a wound contraction inhibitor fortreating scar after the burn, hypertrophic scar, and keloid.

Skin tissues of such as the scar after a burn, the hypertrophic scar,and the keloid, lack cells and blood vessels, and are abundant withfibrous interstitial tissues, which are produced densely andirregularly, in the process for repairing the damaged tissues.Therefore, such damaged tissues are hard and have a light color. Inaddition, the damaged tissues tend to degrade their functionality andcause wound contraction.

It has been considered that these scars are caused by abnormal activityof the fibroblasts due to such things as infectious diseases,inflammations, and diathesis, and by resultant excessive production ofcollagen fibers in the process for the wound healing. However, the basiccritical causes and mechanisms of the scars have not sufficiently beenproved.

At present, either the surgical therapy after crisis (the secondaryredressement) or the conservative treatment using medicine is mainlyperformed for these treatments. In the conservative treatment,tranilast, which is antiallergic drug, heparin ointment, steroids, andthe like are used. These medicines suppress the infectious diseases andthe inflammations, thereby inhibiting the wound contraction. Also, ithas been observed that cytokine, such as interferon γ and interleukin-1,strongly inhibited wound contraction of the skin tissue in vitro. Theseare presently in the clinical trial.

However, in the conventional surgical therapy, there have been manycases in which recurrence happened and the complete recovery was notachieved. That is, not enough therapeutic effect was achieved. Inaddition, when the complete recovery was not achieved in the surgicaltherapy, it was required to simultaneously perform the mechanicaloppression therapy or conservative therapy. Although the conventionalconservative therapy showed the effect of inhibition as to the woundcontraction of the skin tissues, there were many cases in which completerecovery was not achieved. That is, the conventional conservativetherapy also does not achieve decisive results.

BRIEF SUMMARY OF THE INVENTION

The objective of the present invention is to solve the above problems ofthe prior art, and to provide a pharmaceutical formulation forinhibiting wound contraction that strongly inhibits the woundcontraction.

The first aspect of the present invention is a pharmaceuticalformulation for inhibiting wound contraction that includes secretoryleukocyte protease inhibitor as an active ingredient.

Another aspect of the present invention includes a pharmaceuticalformulation for inhibiting wound contraction that contains the secretoryleukocyte protease inhibitor at a concentration of 1–5000 ng/ml.

The further aspect of the present invention includes a pharmaceuticalformulation for inhibiting wound contraction that contains the secretoryleukocyte protease inhibitor at a concentration of 1–100 ng/ml.

The further aspect of the present invention includes a pharmaceuticalformulation for inhibiting wound contraction in which secretoryleukocyte protease inhibitor is obtained by purifying the supernatant ofcultured solution of oral mucosa epithelial cells.

In the further aspect of the present invention, the supernatant of thecultured solution of oral mucosa epithelial cells is made to adsorbusing heparin affinity chromatography, and to elute the adsorbedfraction with a buffer containing 0.5 M sodium chloride. As a result ofthe elution, the resultant fraction is adsorbed using antihuman SLPIantibody immobilized immunoaffinity chromatography. Subsequently, theadsorbed fraction is eluted with 0.1 M glycine buffer to purify thesecretory leukocyte protease inhibitor.

The pharmaceutical formulation for inhibiting wound contraction of thepresent invention may be prepared as an external preparation. It ispreferable to prepare as an ointment. It is also preferable to includepreservatives in the external preparation.

The pharmaceutical formulation for inhibiting wound contraction of thepresent invention may be prepared as an injection. It is preferable toinclude at least one of a stabilizer, a preservative and an analgesicagent in the injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a graph showing a result of in vitro gel contractionexperiment 1.

FIG. 2 illustrates a graph showing a result of in vitro gel contractionexperiment 2.

FIG. 3 illustrates a graph showing a result of in vitro gel contractionexperiment 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be described below in detail.

A pharmaceutical formulation for inhibiting wound contraction of thepresent invention includes secretory leukocyte protease inhibitor(hereafter referred as “SLPI”) as an active ingredient. The SLPI isepithelial cell secreted protein, which is contained in such as skin,mucosa, blood, saliva, synovial fluid, and mucosa secretory of the humanbody, and is also serine protease inhibitor, which strongly inhibits theprotease activity of human leukocyte elastase, cathepsin G, trypsin, andthe like.

The SLPI has a molecular weight of approximately 12 kD (it may berecognized on the electrophoresis gel in the place of about 15 kD). TheSLPI is a polypeptide consisting of 107 amino acids, has eight disulfidebonds in the molecule, and has a unique three-dimensional structure. Inaddition, the SLPI has signal sequence and no sugar chain in theN-terminal.

The SLPI may be purified, for example, from a supernatant of thecultured solution of oral mucosa epithelial cells. In this case, thesupernatant of the cultured solution is adsorbed using heparin affinitychromatography, and then eluted with 0.5 M sodium chloride buffer. Theresultant fraction may be adsorbed using the antihuman SLPI antibodyimmobilized immunoaffinity chromatography, and then may be purified bythe elution with 0.1 M glycine buffer (pH 2.3).

The purified SLPI is preferably preserved in a buffer solution having acomposition similar to the cultured solution of the oral mucosaepithelial cells or body fluid. More preferably, it is preserved in a100 mM TRIS buffer (pH7.5) containing 10 mM calcium chloride and 0.1%human serum albumin since its composition is simple. In addition, it ispreferable to refrigerate the purified SLPI under the light-shieldedcondition to prevent degeneration and oxidation of the SLPI protein. Itis preferable to use the refrigerated SLPI within one month. It is morepreferable to freeze the purified SLPI (e.g., at −30 to −20° C.). It ispreferable to use the frozen SLPI within three months.

A concentration of SLPI in the pharmaceutical formulation is preferably1–5000 ng/ml. The concentration of 1–100 ng/ml is more preferable sincethe substantial maximum effect of wound contracting inhibition isacquired at 100 ng/ml. When the concentration of SLPI is less than 1ng/ml, sufficient wound contracting inhibition is not achieved. On thecontrary, when the concentration of SLPI exceeds 5000 ng/ml, it is noteconomical since the SLPI is abundantly used.

The pharmaceutical formulation for inhibiting wound contraction of thepresent invention may be prepared as an external preparation. It is morepreferable to prepare the pharmaceutical formulation as an ointment. Theointment may contain SLPI as an active ingredient and a base, and may bea homogeneous semisolid or paste form with an appropriate consistency.In addition, the ointment may preferably contain a preservative. Thepreservative is added to improve the stability of the ointment. Thepreservatives listed in Japanese pharmacopoeia are preferably used. Theconcentration of SLPI in the ointment may preferably be the same as theabove pharmaceutical formulation for inhibiting the wound contraction.

Hydrophobic base, such as white petrolatum, petrolatum, Plastibase,silicones, white ointment, vegetable oil, lard, yellow bees wax, andwhite wax; hydrophilic base, such as macrogol; or non-fatty base, suchas hydrogel, may be preferably used as a base.

The ointment is manufactured by the steps of preparing SLPI, base, and apreservative, as needed, and mixing them well to form a semisolid orpaste. Here, it is necessary to avoid the heating to the utmost sincethe heating impairs the wound contracting inhibition of the SLPIprotein. The ointment of the present invention may be preserved as thesame procedure as the above pharmaceutical formulation for inhibitingwound contraction.

The external preparation is not limited to the ointment. It also may beprepared as pastes, cataplasma, liniments, lotions, or plasters inaccordance with the Japanese pharmacopoeia. Further, it is preferable toapply the pharmaceutical formulation to sterilized cloth, paper, plasticfilm, or the like when it is used as the plaster. When it is affixed tothe wound place, it is more preferable to use collagen film or chitinfilm, which promotes the healing.

The pharmaceutical formulation for inhibiting wound contraction may be aliquid injection containing SLPI as an active ingredient. The injectionmay preferably include at least one of a stabilizer (an antioxidant) forimproving the stability of the injection, a preservative, and ananalgesic agent that alleviates pain during the injection.Alternatively, the injection may include the combination of thestabilizer and the preservative, the combination of the stabilizer andthe analgesic agent, or the combination of the preservative and theanalgesic agent. Further, it is most preferable to include all of thestabilizer, the preservative, and the analgesic agent.

The stabilizers, the preservatives, and the analgesic agents listed inthe Japanese pharmacopoeia are preferably used. In addition, theconcentration of SLPI in the injection may preferably be the same as theabove pharmaceutical formulation for inhibiting wound contraction. Asolution for the injection may preferably be isotonic. In addition, theinjection of the present invention may be preserved as the sameprocedure as the above pharmaceutical formulation for inhibiting woundcontraction.

The above embodiments of the present invention have the followingeffects.

The pharmaceutical formulation for inhibiting wound contraction of thepresent invention, including the external preparation and the injection,contains SLPI as an active ingredient. Therefore, the wound contractionmay be strongly inhibited. In addition, since the SLPI is a protein thatexists everywhere in the human body, it is rare to cause side effects,thereby reducing the patient's burden during the treatment. It is alsopossible that the patient can endure administration at high doses andfor a long period of time. In the meantime, treatment for congenitaltracheal cyst and treatment for fibroid lung that is caused due to theside effect of an anticancer drug are carried out using differentactivity of the SLPI. In these treatments, significant side effectsafter administrating the SLPI have not been reported at present. On thecontrary, interferon γ and interleukin-1, which are considered to beeffective for the treatment of the hypertrophic scar and the keloid,have reported side effects for treating the patient having diseasesother than the hypertrophic scar and the keloid. Therefore, when thesedrugs are used for the treatment of the hypertrophic scar and thekeloid, the possibility of causing side effects appears to beconsiderably high.

EXAMPLES

The present invention will now be described in more detail withreference to the following examples and comparative examples.

(Collection of Oral Mucosa Epithelial Cells and Preparation of theCulture System)

Human oral mucosa epithelial cells were obtained at Department of Dentaland Oral Surgery, Nagoya University School of Medicine, with the consentof the patients. Here, donors are healthy adults and no abnormalitiescould be found in many screening tests against infectious diseases,hemograms and the like.

In the obtention, an area to be obtained was sufficiently sterilizedwith the 10% povidone-iodine solution, and then epithelial cells fromthe donor were obtained. The obtained epithelial cells were soaked inthe culture medium for the transportation in the clean operation, andthen preserved at 4° C. The culture system of obtained oral mucosaepithelial cells was made according to the methods of Rheinwald-Green(Nature, 265, 421–424 (1977)) and Hata et al. (Ann. Plast. Surg. 34,530–538 (1995)).

The culture medium for the transportation contains 1000 U/ml penicillinG (manufactured by Meiji Seika Kaisha, Ltd.) 1 mg/ml streptomycin(manufactured by Sigma), 2.5 μg/ml amphotericin B (manufactured byGibco) and 10% fetal calf serum (manufactured by Filtoron; hereafterreferred as “FCS”) in a Dulbecco's Modified Eagle's Medium (manufacturedby Gibco; hereafter referred as “DMEM”).

(Collection of Supernatant of Cultured Solution from Oral MucosaEpithelial Cells)

When the oral mucosa epithelial cells cultured in the 10 cm Falcondishes (made by Becton Dickinson Labware) reached confluent(approximately 2×10⁶ cells/dish), the cells were sufficiently washedwith the phosphate buffer solution. Subsequently, 10 ml DMEM culturedsolution containing 10 μg/ml heparin (manufactured by Nakalai Tesque)was added to the cells, and the cells were cultured in an incubator (37°C., an atmosphere of 10% CO₂) for 48 hours. The supernatant obtainedfrom the cultured solution was recovered, and passed through a 0.45 μmmembrane filter. A filtrate was stored at 4° C., and used forexperiments within 1 week.

(Preparation of the Human Fibroblast)

Regarding specimens of human normal skin, hypertrophic scar and keloidtissues, residual tissues after the operation were obtained atDepartments of Dental and Oral Surgery and Plastic Surgery, NagoyaUniversity School of Medicine, with the consent of patients. Here, thedonors were found to have no abnormality in the infectious disease testand the blood test. Each of the tissues was cut off in 2 mm×2 mm squarepiece. Fibroblast of each tissue (i.e., normal skin tissue, hypertrophicscar tissue or keloid tissue) was obtained by explant culture from thepiece.

More specifically, each piece was divided into 1 mm cube using scissors,three to five pieces of the cube were separately placed on the 35 mmplastic culture dish (made by Falcon Co.), and then DMEM culturedsolution containing 10% FCS was added to the dish. Subsequently, tissuesin each culture dish were cultured in an incubator (37° C., anatmosphere of 5% CO₂) for five to ten days. As a result, the fibroblastsemigrated from the periphery of the tissues, and adsorbed to the bottomsurface of the culture dish while diffusing.

At the time when emigrated fibroblasts covered entire bottom surface ofthe culture dish, fibroblasts were separated and dispersed using asolution containing 0.05% trypsin (manufactured by Gibco) and 0.02%ethylenediaminetetraacetic acid (EDTA) to carry out the subculture. Thesubculture was repeated to obtain enough fibroblasts necessary forexperiments. Here, the fibroblasts between 5th to 9th passages were usedfor the following experiments.

(Establishment of In Vitro Gel Contraction Experimental System)

In accordance with the method of Bell et al. (Pro. Natl. Acad. Sci., 76,1274–1278 (1979)), in vitro gel contraction experimental system wasestablished as follows. First, 2 ml of type I collagen acidic solutionextracted from cattle corium (manufactured by Koken Co. Ltd.), 0.5 ml ofFCS, and 0.5 ml of 6 fold concentrated Eagle minimum essential medium(manufactured by Nissui Pharmaceutical Co.) were mixed to produce 0.2%of collagen solution (pH 7.3). About 3×10⁵ fibroblasts were suspended inthe resultant collagen solution. The collagen solution was cultured inan incubator (37° C., an atmosphere of 5% CO₂) for five to ten minutes.After the viscosity of the collagen solution was increased and thecollagen solution became the gel state, the solution was well mixed.Subsequently, the gel solution was dispensed into plastic culture disheshaving 35 mm of diameter. Resultant collagen gel in the dishes wascultured in an incubator (37° C., an atmosphere of 5% CO₂) for 2 hours.

Then, the collagen gel was carefully washed in DMEM so that it may notbe destroyed. Subsequently, the collagen gel was incubated in the DMEMcultured solution containing one of various test reagents (37° C., anatmosphere of 5% CO₂). The resultant gel was used for following in vitrogel contraction experiments. In the following experiments, collagen gelcultured in DMEM culture solution containing 10% FCS was used as control(a comparative example).

Regarding the measurement of the gel contraction, the diameter of thecollagen gel was measured every 24 hours. Although the gel contractionwas substantially uniformly generated, its shape is not circular.Therefore, the diameter of each gel was measured at three differentplaces and the average diameter of these values was used.

(Purification and Identification of SLPI)

1) Heparin Affinity Chromatography

Protein component included in 3 liter of supernatant of the culturedsolution of oral mucosa epithelial cells was applied to aheparin-Sepharose CL-6B column (made by Amersham Pharmacia BiotechLtd.), and then successively eluted with 50 mM sodium phosphate buffersolution containing three different kind of salt concentrations (i.e.,0.5 M NaCl, 1.0 M NaCl, and 1.5 M NaCl) to collect the fraction.

Resultant fractions having different salt concentrations were used forin vitro gel experiments (the collagen gel including the normalfibroblasts). As a result, the contraction of the collagen gel using thefraction containing 0.5 M sodium chloride was remarkably inhibited incomparison with the fractions of control and the fractions containingthe other concentrations of sodium chloride.

2) Immunoaffinity Chromatography

2 mg of antihuman SLPI antibody (manufactured by R&D systems Inc.) and 1ml of CNBr-activated Sepharose CL-4 B (manufactured by Pharmacia Co.)were reacted in 100 mM sodium bicarbonate buffer solution containing 0.5M sodium chloride (pH 8.8, 4° C.) for 12 hours. Next, antihuman SLPIantibody immobilized Sepharose was made by blocking remaining activegroups with 1 M ethanolamine (pH 8.0). Subsequently, the excessiveadsorbed protein was washed out with 100 mM sodium bicarbonate buffercontaining 0.5 M sodium chloride (pH 8.0) and 100 mM sodium acetatebuffer containing 0.5 M sodium chloride (pH 4.0). Next, the antibodyimmobilized Sepharose was filled with a column (diameter: 0.5 cm;length: 2.5 cm), and then washed with 50 mM sodium biphosphate buffersolution containing 0.2 M sodium chloride (pH 6.8) to equalize.

After the eluate containing 0.5 M sodium chloride, which was obtained bythe heparin affinity chromatography, was adsorbed to the antihuman SLPIantibody immobilized Sepharose, it was eluted with 0.1 M glycine buffer(pH 2.3) to purify SLPI.

Resultant purified SLPI is used for in vitro gel experiments (collagengel containing the normal fibroblasts). As a result, the contraction ofthe collagen gel was remarkably inhibited compared with the controleluted from the column without anti-human SLPI antibody. The quantativeassay of the purified SLPI was carried out using SLPI ELISA(enzyme-linked immunosorbent assay) kit (made by R&D systems Inc.).

3) Polyacrylamide Gel Electrophoresis

Each fraction eluted from the heparin column was condensed bytrichloroacetic acid precipitation and ethanol precipitation.Subsequently, the sample was developed on the gel using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteinretained on the gel was transferred onto a polyvinylidene fluoride(PVDF) membrane (Immobilon-PSQ made by Millipore), and stained withCoomassie brilliant blue.

As a result, the purified SLPI fraction had only included protein havinga molecular weight of 15 kD. Although the other fractions had includedcontaminants other than the 15 kD protein, the stained amount of the 15kD protein was substantially correspondent to the degree of thecontraction inhibiting activity of the collagen gel.

4) N-Terminal Amino Acid Sequence

After excising the 15 kD protein from the PVDF membrane, amino acidsequence at the N-terminal of the protein was analyzed using ABI494Aprotein sequencer (made by Perkin-Elmer Japan Co., Ltd.). As a result,the deciphered amino acid sequence had 100% homology with a human SLPI.

5) Western Blotting Analysis

The PVDF membrane was analyzed with the antihuman SLPI antibody usingWestern blotting. As a result, only the band corresponding 15 kD proteinwas specifically colored.

6) The Confirmation of SLPI mRNA Expression

Total RNAs of the cultured oral mucosa epithelial cells were extractedusing the guanidine isothiocyanate phenol chloroform method.Subsequently, first-strand cDNAs were synthesized from the total RNAsusing the Superscript preamplification system (made by Gibco). Then, thecDNAs were amplified using the cDNA primer of SLPI by the polymerasechain reaction (PCR) method.

As a cDNA primer of the SLPI, the following primers, which added arestriction enzyme EcoRI site, 5′ primer:

-   -   5′-CAGGTACCACCACCATGAAGTCCAGCGGCCTCTT-3′ SEQ ID NO. 1 and 3′        primer:    -   5′-ATGGTACCTCAAGCTTTCACAGGGGAAAC-3′ SEQ ID NO. 2 were made with        reference to the study reported by ABE et al. (J. Clin. Invest.,        87, 2207–2215 (1991)). Subsequently, 2 μl of the first strand        cDNAs, 5 μl of 10×PCR buffer, and 0.5 μl of 12.5U/100 μl Taq        polymerase (manufactured by Perkin-Elmer) were added to the        primers, and then distilled water was added to bring the final        volume to 50 μl. The resultant solution was used for the        reaction.

The resultant reaction solution was carried out for PCR under thefollowing condition. That is, after incubating the solution at 94° C.for 3 minutes, the degeneration process was performed at 94° C. for 30seconds, the primer annealing process was performed at 55° C. for 30seconds, and the elongation process was performed at 72° C. for 30seconds. These three processes were recognized as one cycle. Totally, 35cycles were carried out. The amplification products of the PCR weresubjected to electrophoresis with 1.5% agarose gel, and stained withethidium bromide. A single band was recognized.

Next, the single band (i.e., the amplification product of the PCR) wasexcised from the agarose gel in advance, cDNA was collected and purifiedusing QIAEX II (made by QIAGEN). The cDNAs were subcloned into EcoRIsite of plasmid vector pUC119.

The subcloned cDNAs were determined by the chain terminator method usingan automated DNA sequencer (Li-Cor LC 4000 made by Li-Cor Co.). When asequence that seems to be a normal sequence was compared with the basesequence of known human SLPI, both sequences were identical. Therefore,it was shown that cultured oral mucosa epithelial cells, which secretecontraction inhibiting active factor of collagen gel in the culturedsolution, express mRNA of SLPI.

The above results 1) to 6) verified that human SLPI inhibits contractionof the collagen gel.

(In Vitro Gel Contraction Experiment 1)

SLPI purified by the immunoaffinity chromatography was added to thecollagen gel containing normal fibroblasts, and then the contraction ofthe collagen gel was periodically observed. SLPI was added to thecollagen gel so that the SLPI concentration in the collagen gel ofexamples 1, 2, and 3 was 1 ng/ml, 20 ng/ml, and 100 ng/ml, respectively.DMEM cultured solution containing 10% of FCS was used as a comparativeexample 1. The result was shown in Table 1.

The results regarding examples 1–3 showed that the contractioninhibiting activity of the collagen gel increases as the concentrationof SLPI increases within the concentration range of 1–100 ng/ml. On thecontrary, when in vitro gel contraction experiment was carried out usingthe sample in which 100 ng/ml of SLPI and antihuman SLPI antibody weresimultaneously added, the contraction inhibiting activity of thecollagen gel was degraded compared with example 3. Therefore, it wasconfirmed that the contraction inhibiting activity of the collagen gelwas resulted from SLPI.

(In Vitro Gel Contraction Experiment 2)

Each of 100 ng/ml of purified SLPI (example 4), 100 U/ml of interleukin1β (comparative example 2), and 1000 U/ml of interferon γ (comparativeexample 3) was added to the associated collagen gel containing normalfibroblasts, respectively. After three days, the diameter of eachcollagen gel was measured, and the contraction inhibiting rate (%) ofeach collagen gel was calculated. The results were shown in FIG. 2.

As shown in FIG. 2, the contraction inhibiting activity of the collagengel of example 4 (SLPI) was remarkably greater than that of interleukin1β (comparative example 2) and interferon γ (comparative example 3).

(In Vitro Gel Contraction Experiment 3)

In vitro gel contraction experiment was carried out using collagen gelcontaining normal fibroblasts, collagen gel containing fibroblastsderived from a hypertrophic scar patient, and collagen gel containingfibroblasts derived from a keloid patient. During the experiment, thecontraction of the collagen gel was periodically observed.

10 ng/ml of transforming growth factor (TGF-β) and 100 ng/ml of purifiedSLPI were added to the collagen gel containing normal fibroblasts(example 5). Also, DMEM cultured solution containing 10% of FCS(comparative example 1) or 10 ng/ml of TGF-β(comparative example 4) wasadded to the collagen gel containing normal fibroblasts, respectively.

100 ng/ml of purified SLPI (example 6), or DMEM cultured solutioncontaining 10% of FCS (comparative example 5) was added to the collagengel containing fibroblasts derived from a hypertrophic scar patient,respectively.

100 ng/ml of purified SLPI (example 7), or DMEM cultured solutioncontaining 10% of FCS (comparative example 6) was added to the collagengel containing fibroblasts derived from a keloid patient, respectively.The results were shown in FIG. 3.

As a result, the collagen gel contraction inhibiting activity of each ofexamples 5–7 was greater than that of an associated comparative example,respectively. These results verified that SLPI displays the excellenttherapeutic effect as wound contraction inhibitor for hypertrophic scarand keloid.

(Confirmation of Viable Cells)

Regarding comparative example 1 and example 3, the number of viablecells in the collagen gel during in vitro gel experiment 1 wasperiodically counted according to the method of Ehrlich et al. (Exp.Cell Res. 164, 154–162 (1986)). More specifically, cultured solution wasremoved from comparative example 1 or example 3 immediately after the invitro gel experiment 1 was started, and every 24 hour, respectively.Subsequently, only the collagen gel was transferred to a 15 mlcentrifuge tube (made by Falcon), and then DMEM containing 0.1%collagenase (manufactured by Wako Chemical) was added into the tube.These centrifuge tubes were shaken at 37° C. for 20 minutes, and lightlycentrifuged to collect cell aggregation. Subsequently, the aggregationwas resuspended in the DMEM, and the number of viable cells (cells/ml)was counted using a hemocytometer. The result was shown in Table 1.

TABLE 1 Cell number (cells/ml) Elapsed After 24 After 48 After 72 timeInitial hours hours hours Com. 1.0 × 10⁵ 8.60 ± 0.40 × 8.33 ± 0.42 ×8.47 ± 0.31 × Example 1 10⁴ 10⁴ 10⁴ Example 3 1.0 × 10⁵ 8.27 ± 0.23 ×8.20 ± 0.35 × 8.07 ± 0.12 × 10⁴ 10⁴ 10⁴

As shown in Table 1, the viable cells were confirmed in example 3 evenwhen the fibroblast contacted SLPI, as in comparative example 1, inwhich no fibroblasts contacted SLPI. Accordingly, it was verified thatthe collagen gel contraction inhibiting activity by SLPI included inexample 3 did not result from the stop of contraction due to theextinction of the fibroblasts.

(Confirmation and Consideration Regarding Effects of the Examples)

Basically, hypertrophic scar and keloid were specific diseases for humanbeing. Therefore, it is very difficult to determine effects of thepharmaceutical formulation for inhibiting wound contraction in animalexperiments. To correctly determine the effects of the wound contractioninhibitor, clinical trial must be required. However, it is remarkablydifficult to evaluate quantitatively in the clinical trial. Therefore,an artificial dermis model, which is produced using human fibroblast andtype I collagen that is a component included in skin, has beenconventionally used for determining the effectiveness for thesediseases.

The artificial dermis model forms the structure similar to a naturalhuman dermis through gel contraction when nutrient components andfibroblasts are homogeneously mixed to type I collagen solution toproduce gel. More specifically, the contraction process thereof is asfollows. Immediately after the gel is produced, collagen fibers, whichrandomly contained in the gel, are rearranged due to the action of thefibroblasts. Subsequently, these fibers are crosslinked. This results inthe gel contraction. The dermis-like structure is now clinically appliedas an artificial skin in domestic and overseas.

The gel contraction of the artificial skin, which forms dermis-likestructure, shows morphologically and pathologically very similarphenomenon as the wound contraction in human body. In addition, sinceincreasing and decreasing of the number of cells is not substantiallyobserved in the gel, the artificial skin reflects physiologicalphenomena of the skin tissue. Accordingly, it is possible to considerthis contraction as wound contraction. In addition, it is also possibleto consider factors that promote the gel contraction as wound healingpromoting factor, and factors that inhibit the gel contraction as woundcontraction inhibiting factor.

In fact, when tranilast or heparin, which is now clinically applied asthe wound contraction inhibitor, or interferon, the effect of which isrecognized in the clinical trial, is added to the artificial dermismodel, the gel contraction is inhibited. Further, the fibroblast growthfactor, which is now in the clinical trial as the wound healingpromoter, is added to the artificial model, the promotion of the gelcontraction is observed. Therefore, it is confirmed that the resultsusing the artificial model are substantially identical with the clinicaleffects.

When the SLPI was added to the cultured solution of the gel contractionsystem and cultured them, significant inhibition for the gel contractionwas observed. In addition, it was verified that this inhibiting effectis stronger than that of interleukin 1β and interferon γ, the effects ofwhich are reported strongest at present. It was also confirmed by thefollowing experiment that this effect was not resulted from theextinction of cells. That is, cells were isolated from the gel, thecontraction of which was inhibited by the SLPI, and the isolated cellswere cultured again while measuring the number of viable cells. As aresult, it was confirmed that the number of viable cells was notchanged, and all cells grew in the recultivation.

Also, the morphological observation of actin filament, tubulin,vinculin, integrin α1, α2 and β1 of the fibroblasts in the gel werecarried out using the fluorescent antibody staining. In themorphological observation, although the fibroblasts in the gel werealive, elongation interference of the cell projection was observed, andthe formation of stress fiber by the actin filament was insufficient.However, it was observed that there gave no influence to the expressionof the tubulin, which is a cytoskeleton component, and vinculin, whichis a lining protein of the actin filament. Regarding the expression ofintegrin α1, α2, and β1, there was no difference between the expressionof integrin α2 and β1. However, the expression of integrin α1, whichincreases in scar tissue and keloid, was inhibited. These resultsindicate that the actual fibroblasts in the hypertrophic scar or keloidtissues develop the abnormal cell projections as well as increase thenumber of cells, compared with the fibroblasts of the normal tissue.Also they show that SLPI inhibits the expression of the integrin α1 thatis one of the causes of abnormal wound contraction in the scar tissuesor keloid, and advantageously acts as wound contraction inhibitor.

Based on the above results, the same experiments were carried by usingthe fibroblasts obtained from the sites of hypertrophic scar and keloid.It was conventionally well known that the artificial dermis model usingthese cells showed stronger gel contraction than that using the normalcell. Therefore, the artificial dermis model using these cells was madeand the gel contraction was observed. It was verified that the gelcontraction was significantly promoted. However, when the SLPI was addedto cultured solution of the artificial dermis model using these cells,the contraction was significantly inhibited. This result verified theeffectiveness of the SLPI.

Considering from the above results, when an ointment and an injectioncontaining the SLPI as an active ingredient is used for the treatment ofhypertrophic scar or keloid, as is like the pharmaceutical formulationfor inhibiting wound contraction of the above examples, the woundcontraction of the wound site is considered to be strongly inhibited.

The present embodiments may be embodied with following modifications.

The epithelial cells of patients with scar after the burn, hypertrophicscar, or keloid are obtained and then cultured. SLPI is purified fromthe supernatant of the cultured solution to prepare the woundcontraction inhibitor. The obtained wound contraction inhibitor may beused for the patient from whom the epithelial cells are collected.

In this case, allergic reaction and inflammatory reaction against thepharmaceutical formulation is surely suppressed.

1. A pharmaceutical formulation for inhibiting keloid and scar, theformulation comprising: 1–100 ng/ml of a secretory leukocyte proteaseinhibitor as an active ingredient.
 2. The pharmaceutical formulation forinhibiting keloid and scar according to claim 1, including saidsecretory leukocyte protease inhibitor at a concentration of 100 ng/ml.3. The pharmaceutical formulation for inhibiting keloid and scaraccording to claim 1, wherein said secretory leukocyte proteaseinhibitor is obtained by purifying the supernatant of cultured solutionof oral mucosa epithelial cells.
 4. The pharmaceutical formulation forinhibiting keloid and scar according to claim 1, wherein saidpharmaceutical formulation is prepared as an external preparationincluding a base and the secretory leukocyte protease inhibitor.
 5. Thepharmaceutical formulation for inhibiting keloid and scar according toclaim 4, further comprising a hydrophobic base and a preservative,wherein said external preparation is prepared as an external ointment.6. The pharmaceutical formulation for inhibiting keloid and scaraccording to claim 1, prepared as an injection including said secretoryleukocyte protease inhibitor as an active ingredient.
 7. Thepharmaceutical formulation for inhibiting keloid and scar according toclaim 6, including any one of a stabilizer, a preservative and ananalgesic agent.
 8. The pharmaceutical formulation for inhibiting keloidand scar according to claim 1, wherein said scar is a hypertrophic scar.